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Label-Free Detection of Nucleic Acids

IP.com Disclosure Number: IPCOM000248025D
Publication Date: 2016-Oct-19
Document File: 3 page(s) / 204K

Publishing Venue

The IP.com Prior Art Database

Abstract

Amplification of nucleic acids using polymerase chain reaction (PCR) or other methods is an essential step in diagnosing genetic diseases, determining microbial infections and human identification. Here we describe the design of a novel label-free nucleic acid amplification detection method and biosensor. This invention describes a novel and universal approach for detection of newly synthesised DNA or RNA in solutions. During incorporation of each nucleotide into the growing newly synthesised chain a proton is removed from the 3'-OH group of the primer and released into solution. Therefore, increasing concentration of protons in the solution will be directly proportional to increasing concentration of synthesised DNA or RNA. However, the increase of the proton concentration in solution will be precisely compensated by an increase of total negative charge on the growing DNA molecules hence the pH of the reaction mix stays at the same level. In order to use the increasing concentration of protons for real-time detection of nucleic acid synthesis we describe the use of a proton selective membrane (for example Nafion (Dupont) or Flemion (Asahi) membranes), which will separate the "reaction" and "detection" compartments (Figure 1). Depending on application, the detection of the protons can be accomplished in real time using pH sensitive microelectrodes, semiconductor detector, silicon sensing chip measurement, REDOX potential or visually using a pH indicating dye. This invention describes a label-free detection method & device for nucleic acid following amplification and has not been reported elsewhere. Thus, the invention described here is applicable to nucleic acid research, genetics, molecular diagnostics and human identification processes.

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Summary

Amplification of nucleic acids using polymerase chain reaction (PCR) or other methods is an essential step in diagnosing genetic diseases, determining microbial infections and human identification. Here we describe the design of a novel label- free nucleic acid amplification detection method and biosensor.

This invention describes a novel and universal approach for detection of newly synthesised DNA or RNA in solutions. During incorporation of each nucleotide into the growing newly synthesised chain a proton is removed from the 3'-OH group of the primer and released into solution. Therefore, increasing concentration of protons in the solution will be directly proportional to increasing concentration of synthesised DNA or RNA. However, the increase of the proton concentration in solution will be precisely compensated by an increase of total negative charge on the growing DNA molecules hence the pH of the reaction mix stays at the same level.

In order to use the increasing concentration of protons for real-time detection of nucleic acid synthesis we describe the use of a proton selective membrane (for example Nafion (Dupont) or Flemion (Asahi) membranes), which will separate the "reaction" and "detection" compartments (Figure 1).

Depending on application, the detection of the protons can be accomplished in real time using pH sensitive microelectrodes, semiconductor detector, silicon sensing chip measurement, REDOX potential or visually using a pH indicating dye.

This invention describes a label-free detection method & device for nucleic acid following amplification and has not been reported elsewhere.

Thus, the invention described here is applicable to nucleic acid research, genetics, molecular diagnostics and human identification processes.

Introduction

Fast and reliable real-time detection of nucleic acid synthesis in a complex reaction mix is essential for many different applications in molecular biology and diagnostics. Current methods of detection based on (i) unspecific detection of newly synthesized nucleic acids using intercalating fluorescent dyes (e.g. ethidium bromide and its derivatives, SYBR Green LC Green & LC Green Plus, ResoLight, EvaGreen, Chromofy & SYTO 9); (ii) sequence-specific detection of the products with fluorescently labelled probes (Taqman Probes, Scorpions, Molecular Beacons); (iii) fluorescent or tubidimetric detection of accumulation of inorganic pyrophosphates (PPi). All of the methods require either fluorescent dye or detection reagents in addition to optical detectors.

The present invention describes sensitive, real-time, label-free detection methods and kits for detection of isothermal, PCR and qPCR based amplification of nucleic

Label-Free Detection of Nucleic Acids



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acids. In the assays of the invention, an increasing concentration of protons is detected at real-time in the presence of a proton-selective membrane.

Intellectual Property and Invention

The present invention describes a novel and universal approach for detection...